RNAi vitamin D conjugates

ABSTRACT

The invention provides non-hormonal vitamin D conjugated to therapeutic RNA compounds that result in the compounds having increased absorption, bioavailability or circulating half-life when compared to non-conjugated forms. The vitamin D targeting groups are coupled to the therapeutic RNA compounds via the third carbon on the vitamin D backbone.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 9, 2015, isnamed XTND008US1_SL.txt and is 11,003 bytes in size.

FIELD OF THE INVENTION

The invention provides non-hormonal vitamin D conjugated to therapeuticRNA compounds that result in the compounds having increased absorption,bioavailability or circulating half-life when compared to non-conjugatedforms. The vitamin D targeting groups are coupled to the therapeutic RNAcompounds via the third carbon on the vitamin D backbone.

BACKGROUND OF THE INVENTION

The invention relates to improving the potency, absorption orpharmacokinetic properties of therapeutic RNA compounds to certainvitamin D forms. Vitamin D plays a role in calcium, phosphate, and bonehomeostasis. The hormonal activity of vitamin D is mediated throughbinding to the vitamin D receptor (VDR). It enters the nucleus where itbinds to the vitamin D receptor element (VDRE) present in the promotersof a subset of genes that are thus responsive to hormonal Vitamin D.

Vitamin D is a group of fat-soluble secosteroids. Several forms(vitamers) of vitamin D exist. The two major forms are vitamin D₂ orergocalciferol, and vitamin D₃ or cholecalciferol. Vitamin D without asubscript refers to vitamin D₂, D₃ or other forms known in the art. Inhumans, vitamin D can be ingested as cholecalciferol (vitamin D₃) orergocalciferol (vitamin D₂). The major source of vitamin D for mosthumans is sunlight. Once vitamin D is made in the skin or ingested, itneeds to be activated by a series of hydroxylation steps, first to25-hydroxyvitamin D (25(OH)D₃) in the liver and then to1,25-dihydroxyvitamin D₃ (1α,25(OH)2D₃) in the kidney. 1α,25(OH)2D₃ isthe active “hormonal” form of vitamin D because it binds to VDR.25(OH)D₃ is the “non-hormonal” form of vitamin D and is the majorcirculating form in the human body. It binds the vitamin D BindingProtein (DBP). It is only converted to the hormonal form as needed. Anexample of a non-hormonal vitamin D form is one that lacks a 1α-hydroxylgroup. Non-hormonal vitamin D forms have a greatly reduced affinity forVDR and a greatly increased affinity for DBP.

DBP is the principal transporter of vitamin D metabolites. Itsconcentration in the plasma is 6-7 μM and has been detected in all fluidcompartments. DBP concentrations exceed the physiological vitamin Dmetabolite concentrations. DBP is important for the translocation ofvitamin D from the skin into circulation, and across cell membranes intothe cytoplasm where vitamin D is activated into the hormonal form. Theaffinity of non-hormonal Vitamin D for DBP is significantly higher thanthe affinity of the hormonal form. In contrast, the affinity of thehormonal form to VDR is significantly than the non-hormonal form.

Vitamin D and vitamin D analogs have been approved for the treatment ofosteoporosis and secondary hyperparathyroidism. Vitamin D has also beenshown to inhibit proliferation and induce differentiation in normal aswell as cancer cells. The level of vitamin D required for this activitycauses severe toxicity in the form of hypercalcemia. Analogs of vitaminD have been approved for the treatment of psoriasis and others arecurrently being tested for cancer treatment. Many of the analogsdiscovered to have a reduced calcemic effect contain side-chainmodifications. These modifications do not greatly affect VDR binding,and thus, in cell-based proliferation assays, show equal or evenincreased efficacy. It was shown, however, that many of thesemodifications reduce binding to DBP and thereby reduce the half-life inthe bloodstream.

The addition of poly(ethylene glycol) or (PEG) is a known method ofincreasing the half-life of some compounds by reducing kidney clearance,reducing aggregation, and diminishing potentially unwanted immunerecognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25:403-447(2008)). The PEG is typically used at a considerably large size (20-40kDa) to maximize the half-life in circulation. This can be accomplishedby using either a single large PEG or multiple smaller PEGs attached tothe compound. (Clark et al. J. Biol. Chem. 271:21969-21977 (1996);Fishburn, J. Pharm. Sci. 97:4167-4183 (2008)).

Absorption is a primary focus in drug development and medicinalchemistry because a drug must be absorbed before any medicinal effectscan take place. A drug's absorption profile can be affected by manyfactors. Additionally, the absorption properties of therapeutic RNAcompounds may vary from compound to compound. Alternate routes ofadministration such as intravenous, subcutaneous, or intramuscularinjections are routinely used for some of compounds; however, theseroutes often result in slow absorption and exposure of the therapeuticcompounds to enzymes that can degrade them, thus requiring much higherdoses to achieve efficacy.

RNA interference (RNAi) is a process where RNA molecules inhibit geneexpression often by causing specific mRNA molecules to degrade. Twotypes of RNA molecules—microRNA (miRNA) and small interfering RNA(siRNA)—are central to RNA interference. They bind to the target mRNAmolecules and either increase or decrease their activity. RNAi helpscells defend against parasitic nucleic acids such as those from virusesand transposons. RNAi also influences development.

sdRNA molecules are a class of asymmetric siRNAs comprising a guide(antisense) strand of 19-21 bases. They contain a 5′ phosphate, 2′Ome or2′F modified pyrimidines, and six phosphotioates at the 3′ positions.They also contain a sense strand containing 3′ conjugated sterolmoieties, 2 phospotioates at the 3′ position, and 2′Ome modifiedpyrimidines. Both strands contain 2′ Ome purines with continuousstretches of unmodified purines not exceeding a length of 3. sdRNA isdisclosed in U.S. Pat. No. 8,796,443, incorporated herein by referencein its entirety.

Initial medical applications for RNAi involve genetic diseases such asmacular degeneration and Huntington's disease. Additional applicationsmay include certain cancers, respiratory syncytial virus, herpes simplexvirus type 2, HIV, hepatitis A and B, influenza, and measles.

It remains difficult to deliver RNAi to target tissues, and inparticular, tissues deep within the body. siRNA molecules have a shortin vivo half-life due to endogenous nucleases. Also, targeting specifictissues is challenging. One approach has been high dosage levels ofsiRNA to ensure the tissues have been reached. With these approaches,however, hepatotoxicity was reported.

Therapeutic oligonucleotides, while promising, suffer from a shortplasma half-life as well as from problems with delivery and cellularuptake. Conjugation of oligonucleotides to small molecules has beenproposed to overcome these problems but have not yet been successful.

SUMMARY OF THE INVENTION

The invention provides carrier-drug conjugates comprising a targetinggroup that is non-hormonal vitamin D, an analog, or metabolite thereoflinked at the carbon 3 position to a therapeutic RNA compound. In someembodiments, the non-hormonal vitamin D molecules are not hydroxylatedat the carbon 1 position. The carriers enhance the absorption,stability, half-life, duration of effect, potency, or bioavailability ofthe therapeutic RNA compounds. Optionally, the carriers further comprisescaffolding moieties that are non-releasable such as PEG and othersdescribed in this disclosure.

Thus, the invention provides a carrier-drug conjugate comprising atargeting group that is a non-hormonal vitamin D, analog, or metabolitethereof conjugated to a therapeutic RNA compound at the carbon 3position of the non-hormonal vitamin D targeting group. In a preferredembodiment, the non-hormonal vitamin D is not hydroxylated at the carbon1 position. In another preferred embodiment, the targeting group isconjugated to the therapeutic RNA compound via a scaffold that isselected from the group consisting of poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, a water-solublepolymer, a small carbon chain linker, and an additional therapeuticpeptide.

The invention provides a pharmaceutical composition comprising acarrier-drug conjugate comprising a targeting group that is anon-hormonal vitamin D, analog, or metabolite thereof conjugated via ascaffold at the carbon 3 position to a therapeutic RNA compound having anucleic acid sequence with at least a 90% sequence identity to SEQ IDNO:1, 2, 3, or 4. In a preferred embodiment, the carrier increases theabsorption, bioavailability, or half-life of said therapeutic RNAcompound in circulation. In another preferred embodiment, thenon-hormonal vitamin D is not hydroxylated at the carbon 1 position.

In another embodiment, the scaffold is selected from the groupconsisting of poly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic peptide. In a mostpreferred embodiment, the scaffold is poly(ethylene glycol).

The invention provides a method of treating a patient in need of atherapeutic RNA compound, comprising administering an effective amountof any of the pharmaceutical compositions described herein. In apreferred embodiment, the pharmaceutical composition is delivered tosaid patient by a transdermal, oral, parenteral, subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intrasynovial, intrasternal, intrathecal, intralesional, intracranialinjection, infusion, inhalation, ocular, topical, rectal, nasal, buccal,sublingual, vaginal, or implanted reservoir mode.

The invention provides the use of any of the pharmaceutical compositionsdescribed herein for the manufacture of a medicament for the treatmentof a patient in need of said medicament.

The invention provides a method of manufacturing any of thepharmaceutical compositions described herein comprising conjugating atargeting group and a therapeutic RNA compound, wherein the conjugatingstep utilizes a coupling group. In some embodiments, the coupling groupis selected from the group consisting of an amine-reactive group, athiol-reactive group, a maleimide group, a thiol group, an aldehydegroup, an NHS-ester group, a haloacetyl group, an iodoacetyl group, abromoacetyl groups, a SMCC group, a sulfo SMCC group, a carbodiimidegroup, bifunctional cross-linkers, NHS-maleimido, and combinationsthereof. In other embodiments, the invention provides the pharmaceuticalcompositions resulting from the method described herein, wherein thecompositions comprise a carrier-drug compound containing a linkageselected from the group consisting of a thiol linkage, an amide linkage,an oxime linkage, a hydrazone linkage, and a thiazolidinone linkage. Inanother embodiment, the conjugating step is accomplished bycycloaddition reactions.

The invention provides a pharmaceutical carrier comprising a formula I:B-(L)^(a)-S-(M)^(b)-C   IWherein:B is a targeting group that is a non-hormonal vitamin D, analog, ormetabolite thereof conjugated at the carbon 3 position to L¹;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, polylactic acid, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic moiety;C is an amine-reactive group, a thiol-reactive group, a maleimide group,a thiol group, a disulfide group, an aldehyde group, an NHS-ester group,a 4-nitrophenyl ester, an acylimidazole, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-Maleimido or combinations thereof;L¹ and L² are linkers independently selected from —(CH₂)_(n)—, —C(O)NH—,—HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂— and —NH—;L³ is —(CH₂)_(o)—;n is an integer from 0-3; ando is an integer from 0-3.

In a preferred embodiment, the pharmaceutical carrier comprises formulaV:

In another preferred embodiment, the pharmaceutical carrier comprisesformula VI:

In another preferred embodiment, the pharmaceutical carrier comprisesformula VII:

The invention provides a pharmaceutical composition, comprising atherapeutic RNA compound, a stably attached scaffold, a targeting groupthat is a non-hormonal vitamin D, analog, or metabolite thereofconjugated at the carbon 3 position, wherein after administration to afirst test subject, the therapeutic RNA compound has a half lifemeasured by qRT-PCR or other methods of blood samples taken at aplurality of time points that is greater than a half life of saidtherapeutic RNA compound administered to a second test subject withoutsaid stably attached scaffold moiety and targeting group as measured bythe qRT-PCR or other methods of blood samples taken at said plurality oftime points. In a preferred embodiment, the administration to said firstand second subjects is accomplished by subcutaneous injection.

In another preferred embodiment, the therapeutic RNA compound stablyattached to the scaffold and targeting group retains about the sameactivity as the therapeutic RNA compound not stably attached to saidscaffold and targeting group as measured by a functional assay.

In another embodiment, a scaffold mass range is selected from the groupconsisting of 100 Da. to 20,000 Da., 200 Da. to 15,000 Da., 300 Da. to10,000 Da., 400 Da. to 9,000 Da., 500 Da. to 5,000 Da., 600 Da. to 2,000Da., 1000 Da. to 200,000 Da., 20.00 Da. to 200,000 Da., 100,000 to200,000 Da., 5000 Da. to 100,000 Da., 10,000 Da. to 80,000 Da., 20,000Da. to 60,000 Da., and 20,000 Da. to 40,000 Da. In a preferredembodiment, the scaffold is approximately the same mass as thetherapeutic RNA compound.

The invention provides a carrier-drug conjugate comprising a targetinggroup that is vitamin D, an analog, or a metabolite thereofnon-releasably conjugated to a therapeutic RNA compound. In a preferredembodiment, the vitamin D is non-hormonal. In a more preferredembodiment, the non-hormonal vitamin D is not hydroxylated at the carbon1 position. In another preferred embodiment, the therapeutic RNAcompound is conjugated at the carbon 3 position of said non-hormonalvitamin D targeting group. In another preferred embodiment, thetherapeutic RNA compound retains about the same activity as saidtherapeutic RNA compound not conjugated to said targeting group asmeasured by a functional assay. In another preferred embodiment, thetargeting group is conjugated to the therapeutic RNA compound via ascaffold that is selected from the group consisting of poly(ethyleneglycol), polylysine, polyethyleneimine, poly(propyleneglycol), apeptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, anucleic acid, a glycan, a modifying group that contains a reactivelinker, a water-soluble polymer, a small carbon chain linker, and anadditional therapeutic peptide. In a more preferred embodiment, thescaffold is approximately the same mass as the therapeutic RNA compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a Vitamin D-(3)-PEG_(2k)-aldehyde adduct.The carrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) an aldehyde coupling group.

FIG. 2: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a Vitamin D-(3)-PEG_(2k)-maleimide adduct.The carrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) a maleimide coupling group.

FIG. 3: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a Vitamin D-(3)-PEG_(1.3k)-NHS adduct. Thecarrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) an NHS coupling group.

FIG. 4: MAP4K4 mRNA expression was knocked down by MAP4K4-VitDconjugates in a dose-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides carrier-drug conjugates comprising targetinggroups that are non-hormonal vitamin D, vitamin D analogs, or vitamin Dmetabolites. Examples include vitamin D-based molecules that are nothydroxylated at the carbon 1 (C1) position. The carriers are linked totherapeutic RNA compounds at the carbon 3 (C3) position. As disclosedherein, carrier groups are surprisingly effective when non-hormonalvitamin D forms are used and the therapeutic RNA compound is linked tothe Carbon 3 position. While not wishing to be bound by theory, it isbelieved that the hormonal forms of vitamin D are not appropriate forthe carriers described herein because they can be toxic due to theinduction of hypercalcemia. Also, because the hormonal forms bind thevitamin D receptor in cells, they may improperly target the carrier-drugconjugates to undesired cells or tissues. In contrast, non-hormonalvitamin D forms bind the Vitamin D Binding Protein (DBP) and remain incirculation longer.

The carrier molecules are attached to the therapeutic RNA compoundsusing chemistries described herein, described in WO2013172967,incorporated herein in its entirety, or that are otherwise known in theart. The carriers improve the potency, absorption, bioavailability,circulating half-life or pharmacokinetic properties of the therapeuticRNA compounds. In certain embodiments, the carriers further comprisewhat will be described herein as a “scaffold” that acts, among otherthings, as a non-releasable “spacer” between the targeting group and thetherapeutic RNA compound. In other embodiments, the carriers lack ascaffold.

The carriers are designed to be suitable for use in humans and animals.The carriers serve the purpose of improving the pharmacokineticproperties of a biological or chemical entity that is coupled,conjugated, or fused to the carrier. This occurs through the interactionof the targeting group with DBP. It can actively transport moleculesquickly and effectively from the site of administration to thecirculating plasma, thereby reducing exposure of the drug to degradativeenzymes. The carriers, by binding to DBP, also improve the circulatinghalf-life of the drug. This increases the potency and therapeuticefficacy of the drug by preventing kidney filtration and otherdegradation processes.

The impact on patient health of this new class of therapies will beprofound. A large number of diseases may benefit from RNAi treatment.These include genetic diseases such as macular degeneration andHuntington's Disease. Additionally, certain cancers, liver diseases, andinfectious diseases including respiratory syncytial virus, herpessimplex virus type 2, HIV, hepatitis A and B, influenza, and measles maybenefit from RNAi treatment.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

The term “absorption” is the movement of a drug into the bloodstream. Adrug needs to be introduced via some route of administration (e.g. oral,topical or dermal) or in a specific dosage form such as a tablet,capsule or liquid.

An “antagonist” refers to a molecule capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with the activities of aparticular or specified protein, including its binding to one or morereceptors in the case of a ligand, or binding to one or more ligands incase of a receptor.

“Antibodies” (Abs) and “immunoglobulins” (Igs) refer to glycoproteinshaving similar structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that generally lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Aptamers” are nucleic acid-based compounds that have been selected tobind a specific target. An example of an aptamer-based therapeuticcompound can be found in WO07/035922, incorporated by reference hereinin its entirety.

The term “bioavailability” refers to the fraction of an administereddose of unchanged drug that reaches the systemic circulation, one of theprincipal pharmacokinetic properties of drugs. When a medication isadministered intravenously, its bioavailability is 100%. When amedication is administered via other routes (such as orally), itsbioavailability generally decreases (due to incomplete absorption andfirst-pass metabolism) or may vary from patient to patient.Bioavailability is an important parameter in pharmacokinetics that isconsidered when calculating dosages for non-intravenous routes ofadministration.

“Carriers” are compounds that can be conjugated to, fused to, coupled toor formulated with therapeutic RNA compounds to improve the absorption,half-life, bioavailability, pharmacokinetic or pharmacodynamicproperties of the drugs. They comprise a targeting group, a couplinggroup, and optionally, a scaffold moiety. In some embodiments, carriersmay carry therapeutic RNA compound from the site of subcutaneousinjection into circulation as well as carry the therapeutic RNA compoundin circulation for an extended period of time.

An “effective amount” refers to an amount of therapeutic RNA compoundthat is effective, at dosages and for periods of time necessary, toachieve the desired therapeutic or prophylactic result. A“therapeutically effective amount” of a therapeutic RNA compound mayvary according to factors such as the disease state, age, sex, andweight of the individual. A therapeutically effective amount may bemeasured, for example, by improved survival rate, more rapid recovery,or amelioration, improvement or elimination of symptoms, or otheracceptable biomarkers or surrogate markers. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of thetherapeutic RNA compound are outweighed by the therapeuticallybeneficial effects. A “prophylactically effective amount” refers to anamount of therapeutic RNA compound that is effective, at dosages and forperiods of time necessary, to achieve the desired prophylactic result.Typically, but not necessarily, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

“Half-life” is a scientific term known in the art that refers to theamount of time that elapses when half of the quantity of a test moleculeis no longer detected. An in vivo half-life refers to the time elapsedwhen half of the test molecule is no longer detectable in circulatingserum or tissues of a human or animal.

“Homologs” are bioactive molecules that are similar to a referencemolecule at the nucleotide sequence, peptide sequence, functional, orstructural level. Homologs may include sequence derivatives that share acertain percent identity with the reference sequence. Thus, in oneembodiment, homologous or derivative sequences share at least a 70percent sequence identity. In a preferred embodiment, homologous orderivative sequences share at least an 80 or 85 percent sequenceidentity. In a more preferred embodiment, homologous or derivativesequences share at least an 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99 percent sequence identity. Homologous or derivativenucleic acid sequences may also be defined by their ability to remainbound to a reference nucleic acid sequence under high stringencyhybridization conditions. Homologs having a structural or functionalsimilarity to a reference molecule may be chemical derivatives of thereference molecule. Methods of detecting, generating, and screening forstructural and functional homologs as well as derivatives are known inthe art.

“Hybridization” generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel et al,Current Protocols in Molecular Biology, Wiley Interscience Publishers(1995).

An “individual,” “subject” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, primates (including human and non-human primates) androdents (e.g., mice, hamsters, guinea pigs, and rats). In certainembodiments, a mammal is a human. A “control subject” refers to ahealthy subject who has not been diagnosed as having a disease,dysfunction, or condition that has been identified in an individual,subject, or patient. A control subject does not suffer from any sign orsymptom associated with the disease, dysfunction, or condition.

A “medicament” is an active drug that has been manufactured for thetreatment of a disease, disorder, or condition.

“Morpholinos” are synthetic molecules that are non-natural variants ofnatural nucleic acids that utilize a phosphorodiamidate linkage,described in U.S. Pat. No. 8,076,476, incorporated by reference hereinin its entirety.

“Nucleic acids” are any of a group of macromolecules, either DNA, RNA,or variants thereof, that carry genetic information that may directcellular functions. Nucleic acids may have enzyme-like activity (forinstance ribozymes) or may be used to inhibit gene expression in asubject (for instance RNAi). The nucleic acids used in the inventionsdescribed herein may be single-stranded, double-stranded, linear orcircular. The inventions further incorporate the use of nucleic acidvariants including, but not limited to, aptamers, PNA, Morpholino,antisense, LNA, BNA, PMA, siRNAs and stabilized siRNA, free orconjugated to additional moieties, including sterol, lipids, peptides,GalNAc, or any other moieties introduced for the purpose of facilitationcellular uptake or targeting or other non-natural variants of nucleicacids. By way of example, nucleic acids useful for the invention aredescribed in U.S. Pat. No. 8,076,476, incorporated by reference hereinin its entirety.

“Patient response” or “response” can be assessed using any endpointindicating a benefit to the patient, including, without limitation, (1)inhibition, to some extent, of disease progression, including slowingdown and complete arrest; (2) reduction in the number of diseaseepisodes and/or symptoms; (3) inhibition (i.e., reduction, slowing downor complete stopping) of a disease cell infiltration into adjacentperipheral organs and/or tissues; (4) inhibition (i.e. reduction,slowing down or complete stopping) of disease spread; (5) decrease of anautoimmune condition; (6) favorable change in the expression of abiomarker associated with the disorder; (7) relief, to some extent, ofone or more symptoms associated with a disorder; (8) increase in thelength of disease-free presentation following treatment; or (9)decreased mortality at a given point of time following treatment.

As used herein, the term “peptide” is any peptide comprising two or moreamino acids. The term peptide includes short peptides (e.g., peptidescomprising between 2-14 amino acids), medium length peptides (15-50) orlong chain peptides (e.g., proteins). The terms peptide, medium lengthpeptide and protein may be used interchangeably herein. As used herein,the term “peptide” is interpreted to mean a polymer composed of aminoacid residues, related naturally occurring structural variants, andsynthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally-occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic peptides can besynthesized, for example, using an automated peptide synthesizer.Peptides can also be synthesized by other means such as by cells,bacteria, yeast or other living organisms. Peptides may contain aminoacids other than the 20 gene-encoded amino acids. Peptides include thosemodified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, and are well-known to those of skill in theart. Modifications occur anywhere in a peptide, including the peptidebackbone, the amino acid side chains, and the amino or carboxyl termini.

As used herein, a “pharmaceutically acceptable carrier” or “therapeuticeffective carrier” is aqueous or nonaqueous (solid), for examplealcoholic or oleaginous, or a mixture thereof, and can contain asurfactant, emollient, lubricant, stabilizer, dye, perfume,preservative, acid or base for adjustment of pH, a solvent, emulsifier,gelling agent, moisturizer, stabilizer, wetting agent, time releaseagent, humectant, or other component commonly included in a particularform of pharmaceutical composition. Pharmaceutically acceptable carriersare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants such as ascorbic acid or glutathione, chelating agents, lowmolecular weight proteins or other stabilizers or excipients.

The term “pharmacokinetics” is defined as the time course of theabsorption, distribution, metabolism, and excretion of a therapeutic RNAcompound. Improved “pharmacokinetic properties” are defined as:improving one or more of the pharmacokinetic properties as desired for aparticular therapeutic RNA compound. Examples include but are notlimited to: reducing elimination through metabolism or secretion,increasing drug absorption, increasing half-life, and/or increasingbioavailability.

“PNA” refers to peptide nucleic acids with a chemical structure similarto DNA or RNA. Peptide bonds are used to link the nucleotides ornucleosides together.

“Scaffolds” are molecules to which other molecules can be covalently ornon-covalently attached or formulated. The scaffolds of the inventionmay act as “spacers” between the targeting group and the drug. Spacersare molecular entities that provide physical distance between the twodistinct molecular entities. Scaffolds may also contain a reactive“linker” or may have beneficial therapeutic properties in addition tothe drug. Linkers are the sites of attachment from one molecular entityto another. Thus, the scaffolds of the invention may be, for example,PEG, serum albumin, thioredoxin, an immunoglobulin, a modifying groupthat contains a reactive linker, a water-soluble polymer, or atherapeutic RNA compound. The scaffolds and linkers of the invention arestable (i.e. non-releasable). Non-releasable linkers have more stablechemical bonds than releasable linkers to allow the attached molecularentities to remain attached in vivo. In certain embodiments, however,they may be “releasable” under specific conditions. Releasable linkershave inherent instability and allow for the release of the attachedmolecules under certain conditions over time.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μl/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

The “therapeutic RNA compounds” disclosed herein refer to nucleic acids,and nucleic acid derivatives that are administered to subjects to treatdiseases or dysfunctions or to otherwise affect the health ofindividuals. Non-limiting examples of therapeutic RNA compounds aredrugs that affect metabolic function, analgesics, antipyretics,anti-inflammatory agents, antibiotics, anti-viral compounds, anti-fungalcompounds, cardiovascular drugs, renal drugs, pulmonary drugs, digestivedisease drugs, hematologic drugs, urologic drugs, metabolism drugs,hepatic drugs, neurological drugs, anti-diabetes drugs, anti-cancerdrugs, drugs for treating stomach conditions, drugs for treating colonconditions, drugs for treating skin conditions, and drugs for treatinglymphatic conditions. The term “therapeutic RNA compound” as used hereinhas essentially the same meaning as the terms “drug” or “therapeuticagent.”

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed before or during the course of clinicalpathology. Desirable effects of treatment include preventing theoccurrence or recurrence of a disease or a condition or symptom thereof,alleviating a condition or symptom of the disease, diminishing anydirect or indirect pathological consequences of the disease, decreasingthe rate of disease progression, ameliorating or palliating the diseasestate, and achieving remission or improved prognosis. In someembodiments, methods and compositions of the invention are useful inattempts to delay development of a disease or disorder.

A “vitamin” is a recognized term in the art and is defined as afat-soluble or water-soluble organic substance essential in minuteamounts for normal growth and activity of the body and is obtainednaturally from plant and animal foods or supplements.

“Vitamin D” (also referred to herein as “VitD”) is a group offat-soluble secosteroids. Several forms (vitamers) of vitamin D exist.The two major forms are vitamin D₂ or ergocalciferol, and vitamin D₃ orcholecalciferol. Vitamin D without a subscript refers to vitamin D₂, D₃or other forms known in the art. In humans, vitamin D can be ingested ascholecalciferol (vitamin D₃) or ergocalciferol (vitamin D₂).Additionally, humans can synthesize it from cholesterol when sunexposure is adequate. Cholecalciferol may be modified in the liver or invitro to 25-hydroxycholecalciferol (“25-hydroxy Vitamin D”). In thekidney or in vitro, 25-hydroxy vitamin D can be modified into thedistinct hormonal form of 1,25-hydroxy vitamin D.

“Vitamin D binding protein” or “DBP” is a naturally circulating serumprotein found in all mammals that, among other activities, can bind toand transport vitamin D and its analogs to sites in the liver and kidneywhere the vitamin is modified to its active form, and it retains vitaminD in its various forms in circulation for, on average, 30 days inhumans. A DBP protein sequence is disclosed in SEQ ID NO:5 and anexemplary nucleic acid sequence encoding the DBP protein sequence isdisclosed in SEQ ID NO:6. DBP has multiple naturally-occurring isoforms.Exemplary isoforms are available in the public sequence databases (e.g.Accession Nos. NM_001204306.1, NM_001204307.1, NM_000583.3, BC036003.1,M12654.1, X03178.1, AK223458, P_001191235.1, NP_000574.2, AAA61704.1,AAD13872.1, NP_001191236.1, AAA19662.2, I54269, P02774.1, EAX05645.1,AAH57228.1, AAA52173.1, AAB29423.1, AAD14249.1, AAD14250.1, andBAD97178.1).

The invention contemplates non-hormonal vitamin D conjugates that bindDBP or functional DBP variants and homologs that contain conservative ornon-conservative amino acid substitutions that about retain DBPactivity. DBP binding molecules or functional DBP variants may beidentified using known techniques and characterized using known methods(Bouillon et al., J Bone Miner Res. 6(10):1051-7 (1991), Teegarden et.al., Anal. Biochemistry 199(2):293-299 (1991), McLeod et al, J BiolChem. 264(2):1260-7 (1989), Revelle et al., J. Steroid Biochem.22:469-474 (1985)). The foregoing references are incorporated byreference herein in their entirety.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like.

The invention provides effective routes for administering therapeuticRNA compounds via transdermal, oral, parenteral, subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intrasynovial, intrasternal, intrathecal, intralesional, intracranialinjection, infusion, inhalation, ocular, topical, rectal, nasal, buccal,sublingual, vaginal, or implanted reservoir modes.

In addition, the inventions described herein provide compositions andmethods for maintaining target binding activity, i.e. pharmacodynamics(PD), for therapeutic RNA compounds. It further provides compositionsand methods for improving the pharmacokinetic (PK) profiles oftherapeutic RNA compounds as described herein. The invention furtherprovides compositions and methods for improved drug absorption profilesas compared to the drug absorption profiles for the drugs using the sameroutes of administration or different routes of administration butwithout the inventions described herein. The invention further providescompositions and methods for improved drug bioavailability profiles ascompared to the drug bioavailability profiles for the drugs using thesame routes of administration or different routes of administration butwithout the carriers described herein. The invention further providescompositions and methods for improved drug half-life profiles ascompared to the drug half-life profiles for the drugs using the sameroutes of administration or different routes of administration butwithout the inventions described herein.

The invention also provides alternative routes of drug administrationthat are more cost-effective or favorable to the patients when comparedto the drugs without the inventions described herein.

The non-hormonal vitamin D conjugates disclosed herein may improve theabsorption, half-life, bioavailability, or pharmacokinetic properties ofthe linked therapeutic RNA compounds. While not wishing to be bound bytheory, the carriers have the properties of binding to the body'snatural DBP. DBP may transport the carrier-drug complex from the site ofadministration to the circulating serum. The vitamin D-DBP interactionmay retain the therapeutic RNA compounds in circulation for an extendedperiod of time. This can prevent its excretion from the body andincrease the exposure of the therapeutic RNA compound in the body toachieve a longer lasting therapeutic effect. Additionally, a smallerdose of drug may be required when conjugated the carrier when comparedto the unmodified form.

The therapeutic RNA compound carrier conjugates of the inventiontypically have about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting groupsindividually attached to a therapeutic RNA compound. The structure ofeach of the targeting groups attached to the therapeutic RNA compoundmay be the same or different. In preferred embodiments, one or moretargeting groups are stably or non-releasably attached to thetherapeutic RNA compound at the 5′, 3′ or internal portion of the RNAmolecule. Also contemplated are attachment sites using a combination ofattachment positions.

In another embodiment, the scaffold is a pharmaceutically acceptablecarrier. In preferred embodiments, the scaffold is poly(ethyleneglycol), polylysine, polyethyleneimine, poly(propyleneglycol), apeptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, anucleic acid, a glycan, a modifying group that contain a reactivelinker, a water-soluble polymer, a small carbon chain linker, or anadditional therapeutic moiety.

In one embodiment, water-soluble scaffold moieties have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like.

Peptides carriers can have mixed sequences or be composed of a singleamino acid, e.g., poly(lysine). An exemplary polysaccharide ispoly(sialic acid). An exemplary poly(ether) is poly(ethylene glycol),e.g. m-PEG. □Poly(ethyleneimine) is an exemplary polyamine, andpoly(acrylic) acid is a representative poly(carboxylic acid). Thepolymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein. The polymer backbone can be linear or branched.

Branched polymer backbones are generally known in the art. Typically, abranched polymer has a central branch core moiety and a plurality oflinear polymer chains linked to the central branch core. PEG is commonlyused in branched forms that can be prepared by addition of ethyleneoxide to various polyols, such as glycerol, pentaerythritol andsorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)m in which R represents thecore moiety, such as glycerol or pentaerythritol, and m represents thenumber of arms. Multi-armed PEG molecules, such as those described inU.S. Pat. No. 5,932,462, which is incorporated by reference herein inits entirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as polypropylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), polyvinylpyrrolidone), polylysine,polyethyleneimine, poly(hydroxypropylmethacrylamide), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures thereof. Although themolecular weight of each chain of the polymer backbone can vary, it istypically in the range of about 100 Da to about 100,000 Da.

In other embodiments, the scaffold moiety may be a peptide, serumalbumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid,a glycan, a modifying group that contains a reactive linker, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic compound. In one embodiment, the scaffold moieties arenon-toxic to humans and animals. In another embodiment, the scaffoldsare endogenous serum proteins. In another embodiment, the scaffoldmoieties are water-soluble polymers. In another embodiment, thescaffolds are non-naturally-occurring polymers. In another embodiment,the scaffolds are naturally-occurring moieties that are modified bycovalent attachment to additional moieties (e.g., PEG, polypropyleneglycol), poly(aspartate), biomolecules, therapeutic moieties, ordiagnostic moieties). The scaffolds and linkers of the invention arestable (i.e. non-releasable). In certain embodiments, however, they maybe “releasable” under specific conditions.

The conjugation of hydrophilic polymers, such as PEG, is known in theart. In its most common form, PEG is a linear polymer terminated at eachend with hydroxyl groups: HO—CH2CH2O—(CH2CH2O)n-CH2CH2-OH where ntypically ranges from about 3 to about 4000. In a preferred embodiment,the PEG has a molecular weight distribution that is essentiallyhomodisperse. In another preferred embodiment, the PEG is a linearpolymer. In another preferred embodiment the PEG is a branched polymer.

Many end-functionalized or branched derivatives and various sizes areknown in the art and commercially available. By way of example,conjugation of the PEG or PEO may be carried out using the compositionsand methods described herein and in U.S. Pat. No. 7,803,777 (Defrees etal.) and U.S. Pat. No. 4,179,337 (Davis et al.), each of which areincorporated by reference herein in their entirety.

In some embodiments, smaller therapeutic RNA compounds are paired withsmaller scaffold moieties and larger therapeutic RNA compounds arepaired with larger scaffold moieties. It is contemplated, however, thatsmaller therapeutic RNA compounds could be paired with a larger scaffoldmoiety and vice versa.

In some embodiments, a scaffold that is approximately equal to themolecular weight of a small therapeutic RNA compound results in anefficacious carrier-drug conjugate. Improvements in efficacy may beobtained by empirically adjusting the scaffold size further. Withoutwishing to be bound by theory, the pharmacokinetic properties andefficacy of the conjugates may be enhanced when a scaffold (incombination with linkers as needed) is big enough to ablate potentialsteric hindrance of the drug by DBP binding and vice versa. Thus, atherapeutic RNA compound is conjugated so that its active region isexposed and available for functional activity and the carrier is able tobind DBP. Additional embodiments provide non-releasable attachments thatextend the circulation of therapeutics.

In preferred embodiments, the conjugation of the therapeutic RNAcompound retains about all of its activity following the conjugation.The active region of given therapeutic may be known in the art ordetermined empirically. In other embodiments, the conjugate istherapeutically active while remaining linked to the carrier. Thisembodiment may maximize the time in circulation and as well as itsefficacy.

The scaffolds of the present invention, for example, could have amolecular weight of 100 Daltons (Da.), 500 Da., 1000 Da., 2000 Da., 5000Da., 10,000 Da., 15,000 Da., 20,000 Da., 30,000 Da., 40,000 Da. or60,000 Da. In one embodiment of the invention, “small” scaffolds may bebetween about 100 Da. and 20,000 Da. In another embodiment, “large”scaffolds may be greater than about 20,000 Da. to about 200,000 Da. Inpreferred embodiments, the scaffold moiety is between about 100 Da. and200,000 Da. In more preferred embodiments, the scaffold is between about100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da. and 10,000 Da.,400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da. and 2,000 Da.,1000 Da. and 200,000 Da., 20.00 Da. and 200,000 Da., 100,000 and 200,000Da., 5000 Da. and 100,000 Da., 10,000 Da. and 80,000 Da., 20,000 Da. and60,000 Da., or 20,000 Da. and 40,000 Da. The size of the scaffolds maybe varied to maximize absorption, bioavailability, circulatinghalf-life, or efficacy of the conjugated therapeutic RNA compound.

Another component of the carrier molecule preferably comprises acoupling group that is used to covalently attach the drug to thescaffold or the carrier. The coupling groups of the invention include anamine-reactive group, a thiol-reactive group, a maleimide group, a thiolgroup, an aldehyde group, an NHS-ester group, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-maleimido, combinations thereof, or other coupling groups familiarto persons skilled in the art. The coupling groups of the invention canpromote thiol linkages, amide linkages, oxime linkages, hydrazonelinkages, thiazolidinone linkages or utilize cycloaddition reactionsalso called click chemistry to couple the carrier to a therapeutic RNAcompound. In another embodiment, the composition preferably includes acombination of one or more therapeutic RNA compounds attached to thecoupling group of the scaffold molecule. The linkers of the inventionmay be between about 40 and 100 Daltons. In preferred embodiments, thelinkers may be between about 40-50, 50-60, 60-70, 70-80, 80-90, or90-100 Daltons. The linkers may also be varied to affect the stabilityor releasability of the link between the carrier and the therapeutic RNAcompound.

NHS groups are known to those skilled in the art as being useful forcoupling biomolecules. Utilizing NHS groups allows for flexibility inthe site of carrier conjugation because molecular structure and reactiontime can influence the attachment site and the number of conjugatedcarrier molecules. By way of example, controlling the molar ratio ofNHS-carrier to therapeutic RNA compounds, one skilled in the art canhave some control over the number of carrier molecules attached to thetherapeutic RNA compound. This allows for more than one carrier to beconjugated to a given therapeutic RNA compound.

Conjugation of the carrier to a therapeutic RNA compound is achieved bymixing a solution of the molecules together in a specific molar ratiousing compatible solutions, buffers or solvents. For example, a molarratio of about 1:1, 2:1, 4:1, 5:1, 10:1, 20:1, 25:1, 50:1, 100:1,1000:1, or about 1:2, 1:4, 1:5, 1:10, 1:20 1:25, 1:50, 1:100 or 1:1000of carrier to therapeutic RNA compound could be used. By varying theratio, this could result in different numbers of individual carriersattached to the therapeutic RNA compound, or could help to select aspecific site of attachment. Attachment of the carriers is also pH,buffer, salt and temperature dependent and varying these parametersamong other parameters can influence the site of attachment, the numberof carriers attached, and the speed of the reaction.

Additionally, in order to retain about the same activity of thetherapeutic RNA compounds, conjugation to the carriers will be at a siteon the molecules that do not interfere with therapeutic function.Nucleic acids may require conjugation to the 5′ end, the 3′ end, or aninternal nucleotide, nucleoside, or a derivative thereof. In oneembodiment, the carrier is conjugated to a nucleotide or nucleosideprior to incorporation into a polynucleotide molecule.

In certain embodiments, the present invention provides carriers thatinclude those of formula I:B-(L)^(a)-S-(M)^(b)-C   IWherein:B is a targeting group selected from vitamin D, a vitamin D analog, avitamin D-related metabolite, an analog of a vitamin Drelated-metabolite, a peptide that binds DBP, an anti-DBP antibody, ananti-DBP antibody derivative, a nucleotide aptamer that binds DBP, or asmall carbon-based molecule that binds DBP;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, polylactic acid, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic RNA compound;C is an amine-reactive group, a thiol-reactive group, a maleimide group,a thiol group, a disulfide group, an aldehyde group, an NHS-ester group,a 4-nitrophenyl ester, an acylimidazole, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-maleimido or combinations thereof;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

In preferred embodiments, the present invention provides carriers thatinclude those of formula I:B-(L)^(a)-S-(M)^(b)-C   IWherein:B is a targeting group selected from vitamin D, a vitamin D analog, avitamin D-related metabolite, an analog of a vitamin Drelated-metabolite, or a small carbon-based molecule that binds DBP;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine,poly(propyleneglycol), a peptide, serum albumin, an amino acid, anucleic acid, a glycan, polylactic acid, a water-soluble polymer, or asmall carbon chain linker;C is a maleimide group, a thiol group, a disulfide group, an aldehydegroup, an NHS-ester group, an iodoacetyl group, or a bromoacetyl group;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

In more preferred embodiments, the present invention provides carriersthat include those of formula I:B-(L)^(a)-S-(M)^(b)-C   IWherein:B is a targeting group selected from vitamin D, a vitamin D analog, or avitamin D-related metabolite;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine orpoly(propyleneglycol);C is a maleimide group, a disulfide group, an aldehyde group, anNHS-ester group or an iodoacetyl group;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

In most preferred embodiments, the present invention provides carriersthat include those of formulas IIa, IIb, and IIc:

Wherein:B is a targeting group selected from vitamin D, a vitamin D analog, or avitamin D-related metabolite;S is a scaffold moiety, comprising poly(ethylene glycol), orpoly(propyleneglycol); and C is a maleimide group, a disulfide group, analdehyde group, an NHS-ester group or an iodoacetyl group;L¹ is —(CH₂)_(n)—;L³ is —(CH₂)_(o)—;(M)^(b) are linkers independently selected from —(CH₂)_(n)—, —C(O)NH—,—HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂— and —NH—;b is an integer from 0-4; andn is 3; ando is 1.

In PCT/US2013/031788, which is incorporated herein by reference,conjugation at the C25 position of 25-hydroxy-vitamin D₃ is exemplified.The present invention incorporates conjugation at the C3 position of25-hydroxy-vitamin D₃. This gives improved half-life extension andbioavailability compared to the C25 conjugates.

In certain most preferred embodiments of formula IIa, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVa.

In certain most preferred embodiments of formula IIb, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVb.

In certain most preferred embodiments of formula IIc, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVc.

In certain most preferred embodiment, S is between about 100 Da. and200,000 Da. In other most preferred embodiments, the scaffold moiety isbetween about 100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da.and 10,000 Da., 400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da.and 2,000 Da., 1000 Da. and 200,000 Da., 5000 Da. and 100,000 Da.,10,000 Da. and 80,000 Da., 20,000 Da. and 60,000 Da., or 20,000 Da. and40,000 Da.

In a specific embodiment, the present invention provides a carrierrepresented by formula V.

In another specific embodiment, the present invention provides a carrierrepresented by formula VI.

In another specific embodiment, the present invention provides a carrierrepresented by formula VII.

In certain embodiments, the present invention provides a method forproducing a carrier of formula I:B-(L)^(a)-S-(M)^(b)-C   Icomprising the step of reacting a compound of formula Ia:B-L¹-NH₂   Iawith a compound of formula Ib:HOOC-L³-S-(M)^(b)-C   Ibin the presence of an amide coupling agent,wherein B, S, C and L¹, L³, and (M)^(b) are defined as above and L² is—C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula I. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

One skilled in the art will recognize that any suitable leaving groupmay be coupled with the carboxylic acid of formula Ib in the presence ofa suitable coupling agent to form an active ester of formula Ic:

wherein R is a suitable leaving group including, but are not limited toimidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagentsinclude, but are not limited to 2-chloromethylpyridinium iodide, BOP,PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In some embodiments, thepresent invention provides a method for producing a carrier of formulaI:B-(L)^(a)-S-(M)^(b)-C   Icomprising the step of reacting a compound of formula Ia:B-L¹-NH₂   Iawith a compound of formula Ic:ROOC-L³-S-(M)^(b)-C   Icwherein B, S, C, R and L¹, L³, and (M)^(b) are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, the amide coupling is performed with a base suchas triethylamine or diisopropylethylamine. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

In certain other embodiments, the present invention provides a methodfor producing a carrier of formula IIa:

comprising the steps of reacting a compound of formula Ia:B-L¹-NH₂   Iawith a compound of formula Id:HOOC-L³-S-(M)^(b)-CH₂OH   Idin the presence of an amide coupling agent forming a compound of formulaIe; and

Oxidation of the primary alcohol of formula Ie to an aldehyde of formulaIIa;

wherein B, S, L¹, L³, (M)^(b), b, n and o are defined as above and L² is—C(O)NH— and C is an aldehyde group.

Any suitable oxidizing agent may be used to form a compound of formulaIIa. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula Ie. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

In certain embodiments, any suitable leaving group can be coupled with acarboxylic acid of formula Id in the presence of a suitable couplingreagent to form an active ester of formula If:

wherein R is a suitable leaving group including, but are not limited toimidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagentsinclude, but are not limited to 2-chloromethylpyridinium iodide, BOP,PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

In some embodiments, the present invention provides a method forproducing a carrier of formula Ie:

comprising the step of reacting a compound of formula Ia;B-L¹-NH₂   Iawith a compound of formula If; andROOC-L³-S-(M)^(b)-CH₂OH   If

Oxidation of the primary alcohol of formula Ie to an aldehyde of formulaIIa;

wherein B, S, C, R and L¹, L³, and (M)^(b) are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, the amide coupling is performed with a base suchas triethylamine or diisopropylethylamine. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

Any suitable oxidizing agent may be used to form a compound of formulaIIa. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

In certain other embodiments, the present invention provides a methodfor producing a carrier of formula IIc:

comprising the steps of reacting a compound of formula Ia:B-L¹-NH₂   Iawith a compound of formula Ig:ROOC—S-(M)^(b)-COOH   Igforming a compound of formula Ih; and

Converting a carboxylic acid of formula Ih to an active ester of formulaIIc;

wherein B, S, C, R, L¹, (M)^(b), b, n and o are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable leaving group can be coupled with a carboxylic acid offormula Ih in the presence of a suitable coupling reagent to form anactive ester of formula IIc. Suitable leaving groups include, but arenot limited to imidazole, HOBT, NHS and 4-nitrophenol. Suitable couplingreagents include, but are not limited to 2-chloromethylpyridiniumiodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

In some embodiments, an active ester of formula IIc is formed from acarboxylic acid of formula Ih using a combination of a suitable leavinggroup and a coupling reagent.

In some embodiments, an active ester of formula IIc is formed from acarboxylic acid of formula Ih using a single reagent that produces aleaving group and also effects a coupling reaction. Such reagentsinclude, but are not limited to 1,1′-carbonyldiimidazole,N,N′-disuccinimidyl carbonate, 4-nitrophenyl trifluoroacetate and HBTU.In some embodiments, the single reagent is used alone. In otherembodiments, the single reagent is used with an acyl transfer catalyst.Such acyl transfer catalysts include, but are not limited to DMAP andpyridine. One skilled in the art will recognize that additional acyltransfer catalysts may be used.

In a specific embodiment, the present invention provides a method forproducing a carrier represented by formula V:

comprising the step of reacting a compound of formula Va:

with a compound of formula Vb:

to form a compound of formula Vc;

Reduction of the nitrile group to form the amine of formula Vd;

Reaction of the compound of formula Vd with a compound of formula Ve;

To form a compound of the formula Vf

Oxidation of the primary alcohol of formula Vf to form the aldehyde offormula V.

In some embodiments, the reaction of a compound of formula Vb with acompound of formula Va is promoted by addition of Triton B. One skilledin the art will recognize that other reagents may be used to promotenucleophilic addition to acrylonitrile.

In some embodiments, reduction of the nitrile of formula Vc to the amineof formula Vd is performed using AlCl₃/LAH. One skilled in the art willrecognize that other reduction reagents may be used including sodium,H₂/Pd, H₂/Raney nickel, and diborane.

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, a base such as triethylamine ordiisopropylethylamine is used to promote coupling of the NHS-ester offormula Ve with the amine of formula Vd. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

Any suitable oxidizing agent may be used to form a compound of formulaV. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

In another specific embodiment, the present invention provides a methodfor producing a carrier represented by formula VI:

comprising the steps of reacting a compound of formula Vd:

in the presence of an amide coupling agent with a compound of formulaVIa:

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula VI. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

In another specific embodiment, the present invention provides a methodfor producing a carrier represented by formula VII:

comprising the steps of reacting a compound of formula Vd:

with a compound of formula VIIa:

forming a compound of formula VIIb; and

Converting a carboxylic acid of formula VIIb to an active ester offormula VII;

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, a base such as triethylamine ordiisopropylethylamine is used to promote coupling of the NHS-ester offormula VIIa with the amine of formula Va. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

NHS can be coupled with a carboxylic acid of formula VIIb in thepresence of a suitable coupling reagent to form an active ester offormula VII. Suitable coupling reagents include, but are not limited to2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU, and T3P.

In some embodiments, an active ester of formula VII is formed from acarboxylic acid of formula VIIb using a combination of NHS and acoupling reagent.

In some embodiments, an active ester of formula VII is formed from acarboxylic acid of formula VIIb using a single reagent that produces aleaving group and also effects a coupling reaction. Such reagentsinclude, but are not limited to, N,N′-disuccinimidyl carbonate. In someembodiments, the single reagent is used alone. In other embodiments thereagent is used with an acyl transfer catalyst. Such acyl transfercatalysts include, but are not limited to DMAP and pyridine. One skilledin the art will recognize that additional acyl transfer catalysts may beused.

One skilled in the art will recognize that there are other methods toconjugate a linker and scaffold to the C3 position of vitamin Dderivatives and analogues. For example, the C3 hydroxy group may beacylated by various groups as practiced by N. Kobayashi, K. Ueda, J.Kitahori, and K. Shimada, Steroids, 57, 488-493 (1992); J. G. Haddad, etal., Biochemistry, 31, 7174-7181 (1992); A. Kutner, R. P. Link, H. K.Schnoes, H. F. DeLuca, Bioorg. Chem., 14, 134-147 (1986); and R. Ray, S.A. Holick, N. Hanafin, and M. F. Holick, Biochemistry, 25, 4729-4733(1986). The foregoing references are incorporated by reference in theirentirety. One skilled in the art will recognize that these chemistriescould be modified to synthesize compounds of the formula I:B-(L)^(a)-S-(M)^(b)-C   Iwherein B, S, C, (L)^(a), and (M)^(b) are defined as above.

If desired, therapeutic RNA compound carrier conjugates having differentmolecular weights can be isolated using gel filtration chromatographyand/or ion exchange chromatography. Gel filtration chromatography may beused to fractionate different therapeutic RNA compound carrierconjugates (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer”indicates one targeting group molecule per therapeutic RNA compound,“2-mer” indicates two targeting groups attached to therapeutic RNAcompound, and so on) on the basis of their differing molecular weights(where the difference corresponds essentially to the average molecularweight of the targeting group).

Gel filtration columns suitable for carrying out this type of separationinclude Superdex and Sephadex columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, and (iii) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE).

Separation of therapeutic RNA compound carrier conjugates can also becarried out by reverse phase chromatography using a reverse phase-highperformance liquid chromatography (RP-HPLC) C18 column (AmershamBiosciences or Vydac) or by ion exchange chromatography using an ionexchange column, e.g., a DEAE- or CM-Sepharose ion exchange columnavailable from Amersham Biosciences. The resulting purified compositionsare preferably substantially free of the non-targeting group-conjugatedtherapeutic RNA compound. In addition, the compositions preferably aresubstantially free of all other non-covalently attached targetinggroups.

As described herein, the carriers of the invention may be non-hormonal25-hydroxy vitamin D or analogs thereof having a coupling group on the3′ carbon. “25-hydroxy vitamin D analogs” as used herein includes bothnaturally-occurring Vitamin D metabolite forms as well as otherchemically-modified forms. The carriers of the invention do not includean active (i.e. hormonal) form of Vitamin D (typically having a hydroxylgroup at the 1 carbon). These compounds are based on the vitamin Dstructure and retain partial function of vitamin D (i.e. they interactwith DBP), albeit at varying affinities. The following list exemplifiesvitamin D analog forms known in the art. They may, however, be hormonalor have the C1 hydroxyl group. They are presented here solely for theirchemical properties as vitamin D analogs, not for their functionalhormonal properties: OCT, a chemically synthesized of 1,25(OH)₂ with anoxygen atom at the 22 position in the side chain (Abe et.al., FEBS Lett.226:58-62 (1987)); Gemini vitamin D analog,1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol(BXL0124) (So et al., Mol Pharmacol. 79(3):360-7 (2011)); Paricalcitol,a vitamin D₂ derived sterol lacking the carbon-19 methylene group foundin all natural vitamin D metabolites (Slatopolsky et al., Am J. KidneyDis. 26: 852 (1995)); Doxercalciferol (1α-hydroxyvitamin D₂), likealfacalcidol (1α-hydroxyvitamin D₃), is a prodrug which is hydroxylatedin the liver to 1α,25(OH)₂D₂. Unlike alfacalcidol, doxercalciferol isalso 24-hydroxylated to produce 1α,24(S)—(OH)₂D₂ (Knutson et al.,Biochem Pharmacol 53: 829 (1997)); Dihydrotachysterol2 (DHT2),hydroxylated in vivo to 25(OH)DHT2, 1,25(OH)2DHT2 (McIntyre et al.,Kidney Int. 55: 500 (1999)), ED-71, and eldecalcitol. See also Erben andMusculoskel, Neuron Interact. 2(1):59-69 (2001) and Steddon et al.Nephrol. Dial. Transplant. 16 (10): 1965-1967 (2001). The foregoingreferences are incorporated by reference in their entirety.

In another embodiment, the carrier further comprises a pharmaceuticallyacceptable scaffold moiety covalently attached to the targeting groupand the therapeutic RNA compound. The scaffold moiety of the carriers ofthe invention does not necessarily participate in but may contribute tothe function or improve the pharmacokinetic properties of thetherapeutic RNA compound. The scaffolds of the invention do notsubstantially interfere with the binding of the targeting group to DBP.Likewise, the scaffolds of the invention do not substantially interferewith structure or function of the therapeutic RNA compound. The lengthof the scaffold moiety is dependent upon the character of the targetinggroup and the therapeutic RNA compound. One skilled in the art willrecognize that various combinations of atoms provide for variable lengthmolecules based upon known distances between various bonds (Morrison,and Boyd, Organic Chemistry, 3rd Ed, Allyn and Bacon, Inc., Boston,Mass. (1977), incorporated herein by reference). Other scaffoldscontemplated by the invention include peptide linkers, protein linkerssuch as human serum albumin or immunoglobulin family proteins orfragments thereof, nucleic acid linkers, small carbon chain linkers,carbon linkers with oxygen or nitrogen interspersed, or combinationsthereof. In preferred embodiments, the linkers are non-releasable orstable.

In further embodiments of the invention, the therapeutic RNA compoundsdefined and/or disclosed herein may be chemically coupled to biotin. Thebiotin/therapeutic RNA compound can then bind to avidin.

RNAi conjugated to the vitamin D carriers of the invention are used totreat both inherited and infectious diseases. In preferred embodiments,the conjugates are used to treat, for example, blood conditions, liverconditions, cardiovascular conditions, hepatitis, eye conditions,metabolic conditions, graft rejections, cancer, autoimmune conditions,amyloidosis, and nervous system conditions. In preferred embodiments,vitamin D-RNAi conjugates that have incorporated GalNac, a sugar, fortreating liver conditions. GalNac enhances liver uptake from circulationand thus enhances targeting vitamin D-RNAi to the liver.

Some aspects of the assembly of carriers utilizes chemical methods thatare well-known in the art. For example, Vitamin E-PEG is manufactured byEastman Chemical, Biotin-PEG is manufactured by many PEG manufacturerssuch as Enzon, Nektar and NOF Corporation. Methods of producing PEGmolecules with some vitamins and other therapeutic compounds linked tothem follow these and other chemical methods known in the art. Theattachment of PEG to an oligonucleotide or related molecule occurs, forexample, as the PEG2-N-hydroxysuccinimide ester coupled to theoligonucleotide through the 5′ amine moiety. Several coupling methodsare contemplated and include, for example, NHS coupling to amine groupssuch as a lysine residue on a peptide, maleimide coupling to sulfhydrylgroup such as on a cysteine residue, iodoacetyl coupling to a sulfhydrylgroup, pyridyldithiol coupling to a sulfhydryl group, hydrazide forcoupling to a carbohydrate group, aldehyde for coupling to theN-terminus, or tetrafluorophenyl ester coupling that is known to reactwith primary or secondary amines. Other possible chemical couplingmethods are known to those skilled in the art and can be substituted. Byway of example, conjugation using the coupling groups of the inventionmay be carried out using the compositions and methods described inWO93/012145 (Atassi et al.) and also see U.S. Pat. No. 7,803,777(Defrees et al.), incorporated by reference herein in their entirety.

Exemplary drug formulations of the invention include aqueous solutions,organic solutions, powder formulations, solid formulations and a mixedphase formulations.

Pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, and pharmaceutically acceptablesalts thereof, with any pharmaceutically acceptable carrier, adjuvant orvehicle. Pharmaceutically acceptable carriers, adjuvants and vehiclesthat may be used in the pharmaceutical compositions of this inventioninclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Pharmaceutically acceptable salts retain the desired biological activityof the therapeutic composition without toxic side effects. Examples ofsuch salts are (a) acid addition salts formed with inorganic acids, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like/and salts formed with organic acids suchas, for example, acetic acid, trifluoroacetic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tanic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid and the like; (b) base additionsalts or complexes formed with polyvalent metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium, and the like; or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethlenediamine; or (c) combinations of(a) and (b), e.g. a zinc tannate salt and the like.

The pharmaceutical compositions of this invention may be administered bysubcutaneous, transdermal, oral, parenteral, inhalation, ocular,topical, rectal, nasal, buccal (including sublingual), vaginal, orimplanted reservoir modes. The pharmaceutical compositions of thisinvention may contain any conventional, non-toxic,pharmaceutically-acceptable carriers, adjuvants or vehicles. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, and intracranial injection or infusiontechniques.

Also contemplated, in some embodiments, are pharmaceutical compositionscomprising as an active ingredient, therapeutic RNA compounds describedherein, or pharmaceutically acceptable salt thereof, in a mixture with apharmaceutically acceptable, non-toxic component. As mentioned above,such compositions may be prepared for parenteral administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops, evaporating solutions or aerosols; for inhalation,particularly in the form of liquid solutions or dry powders withexcipients, defined broadly; for transdermal administration,particularly in the form of a skin patch or microneedle patch; and forrectal or vaginal administration, particularly in the form of asuppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example, as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa.(1985), incorporated herein by reference in its entirety. Formulationsfor parenteral administration may contain as excipients sterile water orsaline alkylene glycols such as propylene glycol, polyalkylene glycolssuch as polyethylene glycol, saccharides, oils of vegetable origin,hydrogenated napthalenes, serum albumin or other nanoparticles (as usedin Abraxane™, American Pharmaceutical Partners, Inc. Schaumburg, Ill.),and the like. For oral administration, the formulation can be enhancedby the addition of bile salts or acylcarnitines. Formulations for nasaladministration may be solid or solutions in evaporating solvents such ashydrofluorocarbons, and may contain excipients for stabilization, forexample, saccharides, surfactants, submicron anhydrous alpha-lactose ordextran, or may be aqueous or oily solutions for use in the form ofnasal drops or metered spray. For buccal administration, typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

Delivery of modified therapeutic RNA compounds described herein to asubject over prolonged periods of time, for example, for periods of oneweek to one year, may be accomplished by a single administration of acontrolled release system containing sufficient active ingredient forthe desired release period. Various controlled release systems, such asmonolithic or reservoir-type microcapsules, depot implants, polymerichydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermalpatches, iontophoretic devices and alternative injectable dosage formsmay be utilized for this purpose. Localization at the site to whichdelivery of the active ingredient is desired is an additional feature ofsome controlled release devices, which may prove beneficial in thetreatment of certain disorders.

In certain embodiments for transdermal administration, delivery acrossthe barrier of the skin would be enhanced using electrodes (e.g.iontophoresis), electroporation, or the application of short,high-voltage electrical pulses to the skin, radiofrequencies, ultrasound(e.g. sonophoresis), microprojections (e.g. microneedles), jetinjectors, thermal ablation, magnetophoresis, lasers, velocity, orphotomechanical waves. The drug can be included in single-layerdrug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, orvapor style patches, or could utilize patchless technology. Deliveryacross the barrier of the skin could also be enhanced usingencapsulation, a skin lipid fluidizer, or a hollow or solidmicrostructured transdermal system (MTS, such as that manufactured by3M), jet injectors. Additives to the formulation to aid in the passageof therapeutic RNA compounds through the skin include prodrugs,chemicals, surfactants, cell penetrating peptides, permeation enhancers,encapsulation technologies, enzymes, enzyme inhibitors, gels,nanoparticles and peptide or protein chaperones.

One form of controlled-release formulation contains the therapeutic RNAcompound or its salt dispersed or encapsulated in a slowly degrading,non-toxic, non-antigenic polymer such as copoly(lactic/glycolic) acid,as described in the pioneering work of Kent et al., U.S. Pat. No.4,675,189, incorporated by reference herein. The compounds, or theirsalts, may also be formulated in cholesterol or other lipid matrixpellets, or silastomer matrix implants. Additional slow release, depotimplant or injectable formulations will be apparent to the skilledartisan. See, for example, Sustained and Controlled Release DrugDelivery Systems, J R Robinson ed., Marcel Dekker Inc., New York, 1978;and Controlled Release of Biologically Active Agents, R W Baker, JohnWiley & Sons, New York, 1987. The foregoing are incorporated byreference in their entirety.

An additional form of controlled-release formulation comprises asolution of biodegradable polymer, such as copoly(lactic/glycolic acid)or block copolymers of lactic acid and PEG, is a bioacceptable solvent,which is injected subcutaneously or intramuscularly to achieve a depotformulation. Mixing of the therapeutic RNA compounds described hereinwith such a polymeric formulation is suitable to achieve very longduration of action formulations.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be further enhanced by surfactants, such as,for example, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehdryocholic acid,glycodeoxycholic acid, cycledextrins and the like in an amount in therange of between about 0.1 and 15 weight percent, between about 0.5 and4 weight percent, or about 2 weight percent. An additional class ofabsorption enhancers reported to exhibit greater efficacy with decreasedirritation is the class of alkyl maltosides, such as tetradecylmaltoside(Arnold, J J et al., 2004, J Pharm Sci 93: 2205-13; Ahsan, F et al.,2001, Pharm Res 18:1742-46) and references therein, all of which arehereby incorporated by reference.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as Ph. Helv or a similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient that is solid at roomtemperature but liquid at the rectal temperature and therefore will meltin the rectum to release the active components. Such materials include,but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topical transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When formulated for delivery by inhalation, a number of formulationsoffer advantages. Adsorption of the therapeutic RNA compound to readilydispersed solids such as diketopiperazines (for example, Technosphereparticles (Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv2:1097-1106) or similar structures gives a formulation that results inrapid initial uptake of the therapeutic RNA compound. Lyophilizedpowders, especially glassy particles, containing the therapeutic RNAcompound and an excipient are useful for delivery to the lung with goodbioavailability, for example, see Exubera® (inhaled insulin by Pfizerand Aventis Pharmaceuticals Inc.).

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably 0.5 and about 50 mg/kg body weight per day of the activeingredient compound are useful in the prevention and treatment ofdisease. Such administration can be used as a chronic or acute therapy.The amount of drug that may be combined with the pharmaceutical carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 5% to about 95% active compound(w/w). Preferably, such preparations contain from about 20% to about 80%active compound.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, gender, diet, time of administration,rate of excretion, drug combination, the severity and course of aninfection, the patient's disposition to the infection and the judgmentof the treating physician.

The carrier-drug conjugates described herein provide advantages to drugmanufacturers and patients over unmodified drugs. Specifically, thecarrier-drug conjugate or formulation will be a more potent, longerlasting, and require smaller and less frequent dosing. This translatesinto lowered healthcare costs and more convenient drug administrationschedules for patients. The carrier-drug conjugates can also providesubcutaneous or transdermal routes of administration as alternatives tointravenous injection. These routes can be self-administered by patientsand thus improve patient compliance.

In yet another aspect of the invention, the levels of DBP can beincreased as part of the carrier-drug therapy. It has been reported thatestrogen can increase DBP levels (Speeckaert et al., Clinica ChimicaActa 371:33). It is contemplated here that levels of DBP can beincreased by administration of estrogen for more effective delivery ofcarrier-drug conjugates.

In yet another aspect of the invention, it is contemplated that thecarrier can be used to deliver drugs transdermally. Since DBP normallytransports UV activated vitamin D at locations close to the surface ofthe skin, the use of a transdermal delivery system with the carrierbecomes feasible.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner. Inparticular, the compositions and methods disclosed herein function withall non-hormonal forms of vitamin D, including homologs, analogs, andmetabolites thereof. This includes vitamin D₃ as used in the examplesbelow.

EXAMPLES Example 1 Preparation Exemplary Carriers for CouplingTherapeutic RNA Compounds to Non-Hormonal Vitamin D at the C25 Position

Exemplary carriers were prepared containing vitamin D and 2 kDa PEGscaffolds. One exemplary carrier was thiol-reactive and comprisedvitamin D-PEG with a maleimide reactive group at the C25 position.Another exemplary carrier was amine-reactive and comprised vitamin D-PEGwith an NHS-reactive group. These reagents were prepared as described inWO2013172967 (Soliman et al.), incorporated herein by reference in itsentirety.

Example 2 Preparation of an Exemplary Amino-Terminal Reactive Carrierfor Coupling Insulin Peptides to Non-Hormonal Vitamin D at the C3Position

An exemplary amino-terminal reactive carrier was prepared containing analdehyde reactive group connected to the C3 position of vitamin D and a2 kDa PEG scaffold (VitD-(3)-PEG_(2k)-aldehyde). The aldehyde on thecarrier in this example is used to conjugate to a free amino-terminusgroups on morpholinos, peptide/RNA hybrids, and other molecules known inthe art. The synthesis is outlined in FIG. 1.

Briefly,(S,Z)-3-((E)-2-((1R,3aS,7aR)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexanol(compound Va, 20 mg, 0.049 mmol, 1 equiv., purchased from TorontoResearch Chemicals, catalog number C125700, also known as calcifedioland 25-hydroxyvitamin D) was dissolved in a mixture of anhydroustert-butanol and acetonitrile (10:1, 1 mL), cooled to 4° C.Acrylonitrile (26.6 mg, 0.5 mmol, 10 equiv.) was added to it followed byTriton B, 40% aqueous solution, 10 μL). The mixture was stirred at 4° C.for 2.5 h. The reaction was quenched with cold 2% HCl (10 mL), theaqueous phase was extracted with ether (2×10 mL), dried (MgSO₄) andevaporated to obtain the crude product. This material was purified byflash chromatography (TLC, silica gel, 50% ethyl acetate in hexanes)with 5-20% EtOAc/hexanes as eluent to isolate the desired product,3-(((S,Z)-3-((E)-2-((1R,3aS,7aR)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexyl)oxy)propanenitrile,compound V (15 mg, 68%) as a white solid (R_(f) 0.2 silica gel, 40%EtOAc in hexanes). NMR analysis did not show any appreciable amount ofsolvents.

To a solution of aluminum chloride (66 mg, 0.495 mmol) in anhydrousether (2 mL) at 0° C. under argon was added a solution of lithiumaluminum hydride (1M in ether, 19 mg, 0.5 mL, 0.5 mmol) dropwise. Themixture was stirred for 5 min., a solution of compound Vc (15 mg, 0.033mmol) in ether (3 mL) was added to it dropwise, the reaction mixture wasstirred at 0° C. for 5 min and then at room temperature for 1 h. Thereaction was monitored by MS and TLC (silica gel, 10% MeOH/CHCl₃/0.1%NH₄OH). Ethyl acetate (1 mL) and water (1 mL) were added to the reactionmixture followed by 5% NaOH (5 mL). The organic phase was separated, andthe aqueous phase was extracted with ethyl acetate (5 mL) and ether (5mL). The combined organic phases were washed with brine (5 mL), dried(Na₂SO₄) and evaporated on a rotavap to afford the desired amine,(R)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-(3-aminopropoxy)-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylheptan-2-ol,compound Vd (12.5 mg, 82%) as a pale yellow oil. R_(f) 0.2 (silica gel,20% MeOH/DCM/0.2% NH₄OH). The NMR analysis revealed the presence ˜8% ofethyl acetate.

Compound Vd (12.5 mg, 0.0273 mmol, 1 equiv.), compound Ve (hydroxyl PEGNHS ester, MW 2000 with n≅45 where n is the number of repeating CH₂CH₂Ounits, Jenkem Technology USA #A-5076, 43 mg, 0.0216 mmol, 0.8 equiv.)were dissolved in anhydrous dichloromethane (0.1 mL). Triethylamine (12mg, 16 μl, 0.11 mmol, 4 equiv.) was added and the reaction mixture wasstirred for 20 h at room temperature under nitrogen. The sample wasdried under a stream of nitrogen to afford the crude compound Vf, whichwas purified by flash chromatography using 5-10% MeOH/dichloromethane aseluent to isolate the desired product Vf as a white foam (30 mg, 38%).R_(f) 0.4 (silica gel, 10% methanol in dichloromethane). ¹H NMR analysisof the isolated material confirmed its identity and purity.

To a solution of compound Vf (30 mg, 0.0123 mmol, 1 equiv.),tetrapropylammonium perruthenate (1.0 mg, 0.00284, 0.23 equiv.) andN-methylmorpholine-N-Oxide (4.3 mg, 0.0369 mmol, 3equiv.) in 2 mL of drydichloromethane was added powdered 4 A° molecular sieves (500 mg) andthe reaction mixture was flushed with N₂. The reaction flask was coveredwith aluminum foil to avoid light and it was stirred at room temperaturefor 36 h. Since the R_(f) of both starting material and product is sameon TLC (silicagel, 10% MeOH/dichloromethane), formation of the productwas confirmed by examining the ¹HNMR of an aliquot. The reaction mixturewas filtered through the pad of Celite in a pipette with dichloromethane(15 mL) and N₂ pressure. The combined organics were concentrated under aflow of N₂ and dried on high vacuum for 2 h to get 35 mg (100%) of thecrude product TLC (R_(f): 0.3, 10% MeOH/dichloromethane, staining withPMA). A second run of reaction under the exactly same conditions yieldedanother 35 mg of the product. ¹H NMR of the product from both batches issame and hence combined to get 70 mg of compound V,VitD-(3)-PEG_(2k)-aldehyde.

Example 3 Preparation of an Exemplary Thiol-Reactive Carrier forCoupling Therapeutic RNA Compounds to Non-Hormonal Vitamin D at the C3Position

An exemplary thiol-reactive carrier comprising vitamin D with amaleimide reactive group connected to the C3 position of vitamin D(VitD-(3)-PEG_(2k)-maleimide) was prepared. The maleimide on the carrierin this example was used to conjugate to a free thiol on the protein andpeptide in the examples below. The synthesis is outlined in FIG. 2.

Briefly, compound Vd (23 mg, 0.05 mmol, 1 equiv.) prepared as in Example2, compound VIa (Creative Pegworks cat. # PHB-956, MAL-PEG-COOH, 2 kwith n≅45 where n is the number of repeating CH₂CH₂O units, 79 mg,0.0395 mmol, 0.8 equiv.) and 2-chloro-1-methylpyridinium iodide (32 mg,0.125 mmol, 2.5 equiv.) were dissolved in anhydrous dichloromethane (1mL). Triethylamine (20.4 mg, 28 μl, 0.2 mmol, 4 equiv.) was added andthe reaction mixture was stirred for 4 h at room temperature undernitrogen. The reaction mixture was diluted with dichloromethane (20 mL),washed with 5% aqueous citric acid (20 mL), saturated aqueous sodiumbicarbonate (20 mL), and brine (20 mL). The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated at 30° C. The samplewas purified by silica gel (10 g) flash chromatography. The column waseluted with 1-10% MeOH/dichloromethane. Fractions containing pureproduct were combined together and evaporated on a rotavap, whilemaintaining the temperature at 30° C. The sample was dried under astream of nitrogen to afford compound VI, VitD-(3)-PEG_(2k)-maleimide asa brown gum (58 mg, 48%) (R_(f) 0.25, silica gel, 10% methanol indichloromethane). ¹H NMR analysis of the isolated material confirmed itsidentity and purity.

Example 4 Preparation of an Exemplary Amine-Reactive Carrier forCoupling Therapeutic RNA Compounds to Non-Hormonal Vitamin D at the C3Position

An exemplary amine-reactive carrier comprising vitamin D with an NHSreactive group connected to the C3 position of vitamin D(VitD-(3)-PEG_(1.3k)-NHS) was prepared. The NHS on the carrier in thisexample was used to conjugate to a free thiol on the protein and peptidein the examples below. The synthesis is outlined in FIG. 3.

Briefly, compound Vd (20 mg, 0.044 mmol, 1 equiv.) and compound VIIa(Quanta Biodesign cat. #10140, with n=25 where n is the number ofrepeating CH₂CH₂O units, 44 mg, 0.0346 mmol, 0.8 equiv.) were dissolvedin anhydrous dichloromethane (1 mL). Triethylamine (22.0 mg, 31 μl, 0.22mmol, 5 equiv.) was added and the reaction mixture was stirred for 24 hat room temperature under nitrogen. The reaction mixture was dilutedwith dichloromethane (20 mL), washed with 5% aqueous citric acid (20mL), and brine (20 mL). The organic layer was dried over anhydroussodium sulfate, filtered and concentrated while maintaining thetemperature at 30° C. The sample was purified by silica gel (10 g) flashchromatography. The column was eluted with 1-10% MeOH/dichloromethane.Fractions containing pure product were combined together and evaporatedon a rotavap, while maintaining the temperature below 30° C. The samplewas dried under a stream of nitrogen to afford compound VIIb as a browngum (33 mg, 56%) (R_(f) 0.20, silica gel, 10% methanol indichloromethane). ¹H NMR analysis of the isolated material confirmed itsidentity.

Compound VIIb (31 mg, 0.018 mmol, 1 equiv.), N-hydroxysuccinimide (6.3mg, 0.055 mmol, 3 equiv.), and EDCI (8.6 mg, 0.045 mmol, 2.5 eq.) weredissolved in anhydrous THF (2 mL). Triethylamine (7.4 mg, 10 μL, 0.073mmol, 4 equiv.) was added and the reaction mixture was stirred for 24 hat room temperature under nitrogen. The reaction mixture was dilutedwith dichloromethane (20 mL) and washed with 5% aqueous citric acid (20mL), and brine (20 mL). The organic layer was dried over anhydroussodium sulfate, filtered and concentrated while maintaining thetemperature at 30° C. The sample was dried under a stream of nitrogen toafford compound VII, VitD-(3)-PEG_(2k)-NHS, as a brown gum (38.6mg, >100%) (R_(f) 0.25, silica gel, 10% methanol in dichloromethane). ¹HNMR analysis of the isolated material confirmed its identity and purity.

Example 5 Preparation of Inhibitory RNA (RNAi) Coupled to Non-HormonalVitamin D at the C3 Position

In this example, the VitD-(25)-PEG_(2K)-NHS carrier from Example 1 andthe VitD-(3)-PEG_(1.3K)-NHS (Compound VII from Example 4) are conjugatedto siRNA in order to extend the half-life and enhance cell penetrationof the siRNA.

Synthesis of VitD-(2S)-PEG2K-siRNA

RNA oligonucleotides listed in Table 3 were synthesized by TriLinkBioTechnologies (San Diego, Calif.). The oligonucleotides were modifiedto impart resistance to nucleases, contain a 5′-amine modification forcoupling to NHS-activated vitamin D carriers, and contain an optionalCy3 fluorophore for visual tracking and sensitive quantitation of theoligonucleotides. One of the siRNA molecules is complementary tosequences within the MAP4K4 gene. The other is not complementary to anygenomic sequences (negative control).

TABLE 3 Sequence name Gene target 5′-modification 3′-modification NTC2_NNegative control NH₂ None NTC_N_cy3 Negative control NH₂ Cy3 10001_N_0MAP4K4 NH₂ Cy3

The anti-MAP4K4 sdRNA sequences are as follows:

Antisense strand: (SEQ ID NO: 7)PmU.A.G.A.fC.fU.fU.fC.fC.A.mC#A#mG#A#mA#mC#mU#mC#U  Sense strand:(SEQ ID NO: 8) mC.mU.G.mU.G.G.mA.A.G.mU.mC#mU#mA1where “.” is a phosphate atom, “#” is thiophosphate, “m” is 2′Ome, “f”is 2′-Fluoro, “1” is Cholesterol, and “P” is the 5′ phosphate.

Similarly, the negative control (non-targeting control or NTC) sequenceis as follows:

Antisense strand: (SEQ ID NO: 9)PmU.fC.G.fC.G.A.fA.A.fC.A.fU.G.mU.A#A#A#mC#mC#A#A  Sense strand:(SEQ ID NO: 10) mU.mU.A.mC.A.mU.G.mU.mU.mU.mC.G.mC#mG#mA1where “.” is a phosphate atom, “#” is thiophosphate, “m” is 2′Ome, “f”is 2′-Fluoro, “1” is Cholesterol, and “P” is the 5′ phosphate. The basicnucleic acids sequences are in SEQ ID NOs: 1 to 4.

The oligonucleotides bearing a 5′-amine modification (10 mM) were mixedwith the VitD-(25)-PEG2K-NHS carrier (200 mM) in 10 mM HEPES pH=8.5 andallowed to react at room temperature for two hours. The conjugation wasconfirmed by analysis using a 15% TBE-urea PAGE gel with visualizationprovided by SYBR® Gold Nucleic Acid Gel Stain (Life Technologies,catalog # S-11494) or Cy3 fluorescence. The conjugated oligonucleotideswere purified using C18 spin columns (Thermo Scientific Pierce catalog #89870) according to the manufacturer's instructions. Briefly, theconjugates were loaded on the C18 resin in 10% acetonitrile in C18buffer (25 mM HEPES and 25 mM NH4Cl), washed with solution containingincremental increases of acetonitrile up to 20%, and then eluted with50% acetonitrile. The samples were dried under vacuum and analyzed by15% TBE-urea PAGE as above. Oligonucleotide concentrations weredetermined by measuring the UV absorbance at 260 nm and using theextinction coefficient as calculated by the manufacturer (TriLinkBioTechnologies).

Synthesis of VitD-(3)-PEG_(1.3k)-siRNA

The VitD-(3)-PEG_(1.3)K-NHS carrier (compound 21, example 3) isconjugated to amine-modified oligonucleotides listed in Table 3 in asimilar fashion as described above for the VitD-(25)-PEG_(2K)-NHScarrier. The siRNA and scaffolds that may be conjugated at the C3position of a non-hormonal vitamin D are not limited to the particularones in this example.

Activity of siRNA Constructs in Cell-Based mRNA Knockdown Assay:

RNA-carrier conjugates were mixed in equimolar amounts with acomplementary oligonucleotide and annealed to form a double-strandedsiRNA product. 60 mM of each oligonucleotide strand were annealed byfirst heating for 1 minute at 95 degrees C. and then cooled at roomtemperature for one hour.

HeLa cells were trypsinized, counted, and resuspended in EMEM medium(ATCC catalog #:30-2003) containing 6% FBS (Gibco catalog #: 16140071).50 ml of siRNA in 0% FBS EMEM medium was added to each well of a 96-wellplate. 6000 cells in 50 ml of medium were then added to each well. Thefinal concentration of siRNA ranged from 0.4 to 2 mM. Cells and siRNAwere incubated for 48 hours, the medium was removed, and the cells werewashed once with PBS. Total RNA was isolated using Purelink™ Pro 96Total RNA Purification Kit (Ambion catalog #: 12173-011A) according tothe manufacturer's instructions. Total RNA was used undiluted andquantitated with Quanta qScript XLT One-Step RT-qPCR Tough Mix, Rox (VWRcatalog #:89236-672) according to the manufacturer's instructions. Thefollowing TaqMan® probes were used: Human GAPDH Endogenous Control,VIC®-labeled MGB Probe, Primer limited (ABI catalog #4326317E, 150 nMprimers, 250 nM probe) and probe from Human MAP4K4 TaqMan® GeneExpression Assay (TaqMan®, Hs00377405_m1, FAM-labeled, 900 nM primers,250 nM probe).

MAP4K4 mRNA was normalized to the ubiquitous GAPDH mRNA, as well as toexogenously added negative control siRNA (NTC_N_cy3). MAP4K4 mRNA isshown in FIG. 4 as a function of siRNA concentration. Thecy3-MAP4K-PEG_(2K)-(25)-VitD conjugate was superior to the unmodifiedsiRNA, cy3-MAP4K-NH₂, at knocking down mRNA levels of its target geneand shows similar activity as the cy3-MAP4K siRNA. This demonstratesthat conjugation of siRNA to vitamin D results in about the sameactivity as unmodified siRNA, and in some cases superior activity.Imaging of HeLa cells after incubation with cy3-MAP4K-PEG_(2K)-(25)-VitDconfirmed that the fluorescent conjugate was delivered into the interiorof the cells (Data not shown).

EXEMPLARY SEQUENCES (MAP4 Kinase 4 siRNA antisense strand) SEQ ID NO: 1 UAGACUUCCACAGAACUCU (NTC2_N siRNA antisense strand) SEQ ID NO: 2 UCGCGAAACAUGUAAACCAA (MAP4 Kinase 4 siRNA sense strand) SEQ ID NO: 3 CUGUGGAAGUCUA (NTC2_N siRNA sense strand) SEQ ID NO: 4  UUACAUGUUUCGCGA(Vitamin D Binding Protein (DBP)) SEQ ID NO: 5 MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGTFEQVSQFVKEVVSFTEACCAEGADPDCYDTRTSAFSAKSCESNSPFPVHPGTAECCTKEGFERKLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLIKLAQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHLPEVFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNIL (Vitamin D Binding Protein (DBP)) SEQ ID NO: 6 TTTAATAATAATTCTGTGTTGCTTCTGAGATTAATAATTGATTAATTCATAGTCAGGAATCTTTGTAAAAAGGAAACCAATTACTTTTGGCTACCACTTTTACATGGTCACCTACAGGAGAGAGGAGGTGCTGCAAGACTCTCTGGTAGAAAAATGAAGAGGGTCCTGGTACTACTGCTTGCTGTGGCATTTGGACATGCTTTAGAGAGAGGCCGGGATTATGAAAAGAATAAAGTCTGCAAGGAATTCTCCCATCTGGGAAAGGAGGACTTCACATCTCTGTCACTAGTCCTGTACAGTAGAAAATTTCCCAGTGGCACGTTTGAACAGGTCAGCCAACTTGTGAAGGAAGTTGTCTCCTTGACCGAAGCCTGCTGTGCGGAAGGGGCTGACCCTGACTGCTATGACACCAGGACCTCAGCACTGTCTGCCAAGTCCTGTGAAAGTAATTCTCCATTCCCCGTTCACCCAGGCACTGCTGAGTGCTGCACCAAAGAGGGCCTGGAACGAAAGCTCTGCATGGCTGCTCTGAAACACCAGCCACAGGAATTCCCTACCTACGTGGAACCCACAAATGATGAAATCTGTGAGGCGTTCAGGAAAGATCCAAAGGAATATGCTAATCAATTTATGTGGGAATATTCCACTAATTACGGACAAGCTCCTCTGTCACTTTTAGTCAGTTACACCAAGAGTTATCTTTCTATGGTAGGGTCCTGCTGTACCTCTGCAAGCCCAACTGTATGCTTTTTGAAAGAGAGACTCCAGCTTAAACATTTATCACTTCTCACCACTCTGTCAAATAGAGTCTGCTCACAATATGCTGCTTATGGGGAGAAGAAATCAAGGCTCAGCAATCTCATAAAGTTAGCCCAAAAAGTGCCTACTGCTGATCTGGAGGATGTTTTGCCACTAGCTGAAGATATTACTAACATCCTCTCCAAATGCTGTGAGTCTGCCTCTGAAGATTGCATGGCCAAAGAGCTGCCTGAACACACAGTAAAACTCTGTGACAATTTATCCACAAAGAATTCTAAGTTTGAAGACTGTTGTCAAGAAAAAACAGCCATGGACGTTTTTGTGTGCACTTACTTCATGCCAGCTGCCCAACTCCCCGAGCTTCCAGATGTAGAGTTGCCCACAAACAAAGATGTGTGTGATCCAGGAAACACCAAAGTCATGGATAAGTATACATTTGAACTAAGCAGAAGGACTCATCTTCCGGAAGTATTCCTCAGTAAGGTACTTGAGCCAACCCTAAAAAGCCTTGGTGAATGCTGTGATGTTGAAGACTCAACTACCTGTTTTAATGCTAAGGGCCCTCTACTAAAGAAGGAACTATCTTCTTTCATTGACAAGGGACAAGAACTATGTGCAGATTATTCAGAAAATACATTTACTGAGTACAAGAAAAAACTGGCAGAGCGACTAAAAGCAAAATTGCCTGATGCCACACCCACGGAACTGGCAAAGCTGGTTAACAAGCACTCAGACTTTGCCTCCAACTGCTGTTCCATAAACTCACCTCCTCTTTACTGTGATTCAGAGATTGATGCTGAATTGAAGAATATCCTGTAGTCCTGAAGCATGTTTATTAACTTTGACCAGAGTTGGAGCCACCCAGGGGAATGATCTCTGATGACCTAACCTAAGCAAAACCACTGAGCTTCTGGGAAGACAACTAGGATACTTTCTACTTTTTCTAGCTACAATATCTTCATACAATGACAAGTATGATGATTTGCTATCAAAATAAATTGAAATATAATGCAAACCATAAAAAAAAAAAAAAAAAAAAAA A

All publications and patent documents disclosed or referred to hereinare incorporated by reference in their entirety. The foregoingdescription has been presented only for purposes of illustration anddescription. This description is not intended to limit the invention tothe precise form disclosed. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed:
 1. A carrier-drug conjugate comprising a targetinggroup that is a non-hormonal vitamin D conjugated to a therapeutic RNAcompound at the carbon 3 position of said non-hormonal vitamin Dtargeting group.
 2. The carrier-drug conjugate of claim 1, wherein saidnon-hormonal vitamin D is not hydroxylated at the carbon 1 position. 3.The carrier-drug conjugate of claim 1, wherein said targeting group isconjugated to said therapeutic RNA compound via a scaffold that isselected from the group consisting of poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, a water-solublepolymer, a small carbon chain linker, and an additional therapeuticpeptide.
 4. A pharmaceutical composition comprising a carrier-drugconjugate comprising a targeting group that is a non-hormonal vitamin Dconjugated via a scaffold at the carbon 3 position to a therapeutic RNAcompound having a nucleic acid sequence with at least a 90% sequenceidentity to SEQ ID NO:1.
 5. The pharmaceutical composition of claim 4,wherein said carrier increases the cellular uptake, absorption,bioavailability, or half-life of said therapeutic RNA compound incirculation.
 6. The pharmaceutical composition of claim 4, wherein saidnon-hormonal vitamin D is not hydroxylated at the carbon 1 position. 7.The pharmaceutical composition of claim 5, wherein said scaffold isselected from the group consisting of poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, a water-solublepolymer, a small carbon chain linker, and an additional therapeuticpeptide.
 8. The pharmaceutical composition of claim 7, wherein saidscaffold is poly(ethylene glycol).
 9. A carrier-drug conjugatecomprising a targeting group that is vitamin D non-releasably conjugatedto a therapeutic RNA compound, wherein said therapeutic RNA compound isconjugated at the carbon 3 position of said non-hormonal vitamin Dtargeting group.
 10. The carrier-drug conjugate of claim 9, wherein saidvitamin D is non-hormonal.
 11. The carrier-drug conjugate of claim 9,wherein said non-hormonal vitamin D is not hydroxylated at the carbon 1position.
 12. The carrier-drug conjugate of claim 9, wherein saidtherapeutic RNA compound retains about the same activity as saidtherapeutic RNA compound not conjugated to said targeting group asmeasured by a functional assay.
 13. The carrier-drug conjugate of claim9, wherein said targeting group is conjugated to said therapeutic RNAcompound via a scaffold that is selected from the group consisting ofpoly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic peptide.
 14. Thecarrier-drug conjugate of claim 13, wherein said scaffold isapproximately the same mass as said therapeutic RNA compound.